The present application claims priority to German Application No. DE 10042053.2 filed Aug. 26, 2000 and German Application No. DE 10123071.0 filed May 11, 2001, the entire contents of both applications are incorporated herein by reference.
1. Field of the Invention
The invention provides nucleotide sequences from Coryneform bacteria which code for the ccpA2 gene and a process for the fermentative preparation of amino acids, in particular L-lysine, by attenuation of the ccpA2 gene. The ccpA2 gene codes for the CcpA2 protein, which is the catabolite control protein A.
2. Discussion of the Background
L-Amino acids, particularly L-lysine, are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and, most particularly, in animal nutrition.
It is known that amino acids are prepared by fermentation of strains of Coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, attempts are continuously being made to improve the preparation processes. Improvements to the process may concern measures relating to fermentation, for example, stirring and oxygen supply, or the composition of the nutrient media, such as the sugar concentration during the fermentation, or the working up to the product form by, for example, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
The output properties of these microorganisms are improved by employing methods of mutagenesis, selection and mutant selection. These methods yield strains that produce amino acids and are resistant to antimetabolites or are auxotrophic for metabolites important for regulation.
For a number of years, methods of recombinant DNA technology have also been used for improving the L-amino acid-producing strains of Corynebacterium. However, there remains a critical need for improved methods of producing L-amino acids and thus for the provision of strains of bacteria producing higher amounts of L-amino acids. On a commercial or industrial scale even small improvements in the yield of L-amino acids, or the efficiency of their production, are economically significant. Prior to the present invention, it was not recognized that attenuation of the ccpA2 gene encoding the catabolite control protein A (CcpA2) would improve L-amino acid yields.
An object of the present invention is to provide novel measures for the improved production of L-amino acids or amino acid, where these amino acids include L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-arginine and the salts (monohydrochloride or sulfate) thereof.
One object of the present invention is providing a novel process for improving the fermentative production of said L-amino acids, particularly L-lysine. Such a process includes enhanced bacteria, preferably enhanced Coryneform bacteria, which express attenuated amounts of the CcpA2 catabolite control activity.
Thus, another object of the present invention is providing such a bacterium, which expresses an attenuated amount of CcpA2 catabolite control protein or gene products of the ccpA2 gene.
Another object of the present invention is providing a bacterium, preferably a Coryneform bacterium, which expresses a polypeptide that has an attenuated CcpA2 catabolite control activity.
Another object of the invention is to provide a nucleotide sequence encoding a polypeptide which has CcpA2 catabolite control protein sequence. One embodiment of such a sequence is the nucleotide sequence of SEQ ID NO: 1.
A further object of the invention is a method of making CcpA2 catabolite control protein or an isolated polypeptide having a CcpA2 catabolite control activity, as well as use of such isolated polypeptides in the production of amino acids. One embodiment of such a polypeptide is the polypeptide having the amino acid sequence of SEQ ID NO: 2.
Other objects of the invention include methods of detecting nucleic acid sequences homologous to SEQ ID NO: 1, particularly nucleic acid sequences encoding polypeptides that have CcpA2 catabolite control activity, and methods of making nucleic acids encoding such polypeptides.
The above objects highlight certain aspects of the invention. Additional objects, aspects and embodiments of the invention are found in the following detailed description of the invention.